Fusion proteins of HIV regulatory/accessory proteins

ABSTRACT

Nucleic acids are disclosed encoding fusion proteins comprising the amino acid sequence of at least four non-attenuated HIV proteins selected from the group consisting of Vif, Vpr, Vpu, Vpx, Rev, Tat, and Nef, wherein the fusion protein does not contain a specific cleavage sequence for a cellular protease. Also disclosed are vectors comprising the nucleic acids and methods of preparing the fusion protein by transfecting a host cell with the nucleic acids or with the vectors containing the nucleic acids, expressing the fusion proteins, and recovering the fusion proteins.

The invention relates to fusion proteins comprising the amino acidsequence of at least four HIV proteins selected from Vif, Vpr, Vpu, Vpx,Rev, Tat and Nef or derivatives of the amino acid sequence of one ormore of said proteins, wherein the fusion protein is not processed toindividual HIV proteins having the natural N and C termini. Theinvention further concerns nucleic acids encoding said proteins, vectorscomprising said nucleic acids, and methods for producing said proteins.The fusion protein, nucleic acids and vectors are usable as vaccines forthe at least partial prophylaxis against HIV infections.

BACKGROUND OF THE INVENTION

The Human Immunodeficiency virus (HIV) is the causative agent of theAcquired Immunodeficiency Syndrome (AIDS). Like all retroviruses thegenome of the virus encodes the Gag, Pol and Env proteins. In addition,the viral genome encodes further regulatory proteins, i.e. Tat and Rev,as well as accessory proteins, i.e. Vpr, Vpx, Vpu, Vif and Nef.

Despite public health efforts to control the spread of the AIDS epidemicthe number of new infections is still increasing. The World HealthOrganization estimated the global epidemic at 36.1 million infectedindividuals at the end of the year 2000, 50% higher than what waspredicted on the basis of the data a decade ago (WHO & UNAIDS. UNAIDS,2000). Globally, the number of new HIV-1 infections in 2000 is estimatedat 5.3 million.

Given the steady spread of the epidemic, there is still a need to bringan effective vaccine to the clinic. A number of different HIV-1 vaccinedelivery strategies such as novel vectors or adjuvant systems have nowbeen developed and evaluated in different pre-clinical settings as wellas in clinical trials. The first vaccine candidate that entered a phaseIII clinical trial is based on envelope gp 120 protein in alum (Franciset al., AIDS Res. Hum. Retroviruses 1998; 14 (Suppl 3)(5): S325-31). Thephase III trials have been started although the vaccine did not prove tobe too successful in the earlier phase II trial.

Following many years of prophylactic vaccine efforts based on envelopeantigens, more recent efforts have focused on the use of regulatoryproteins such as Tat, Nef and Rev as candidate vaccine antigens. The useof these regulatory antigens in therapeutic settings has been ongoingfor several years (Miller et al., Nature Medicine 1997, 3, 389-94,Calarota et al., Lancet 1998, 351, 1320-5, Ayyavoo et al., AIDS, 2000,14, 1-9). More recently the use of these antigens in prophylacticvaccine studies in small pre-clinical trials has revealed promise. Theuse of Tat and Rev, or Tat alone as a prophylactic vaccine candidate,was demonstrated to control SIVmac (Osterhaus et al., Vaccine 1999, 17,2713-4). Moreover, there are indications that CTL directed towards thevirus early regulatory proteins are important for eliminating infectedcells prior to their high level production of mature virions (van Baalenet al., J. Gen. Virol 1997, 78, 1913-8; Addo et al., PNAS, 2001, 98,1781-6).

Although the regulatory/accessory proteins of HIV induce an effectiveimmune response, most, if not all, of them have serious side effects,which limit up to now their use as vaccine: Nef, Tat and Vpu have beenshown to play a role in the down regulation of CD4+ and/or MHC class Iexpression (Howcroft et al., Science, 1993, 260, 1320-2; Schwartz etal., Nature Med. 1996, 2, 338-42; Swann et al., Virology, 2001, 282,267-77; Janvier et al., J. Virol., 2001, 78, 3971-6, Weissmann et al.,PNAS 1998, 95, 11601-6). It is known that Tat mediates acute immunesuppression in vivo (Cohen et al., PNAS, 1999, 96, 10842-10847).Immunosuppressive effects have also been described for Vpr (Ayyavoo etal., Nature Med., 1997, 3: 1117-1123). It has been described that Vprand Vpx have differential cytostatic and cytotoxic effects in yeastcells (Zhang et al., Virology, 1997, 230, 103-12). Thus, most, if notall accessory/regulatory proteins of HIV seem to have functionalproperties that are not desired in a vaccine formulation.

Attempts to reduce the harmful effects of the HIV proteins are disclosedin WO 02/06303. In particular, WO 02/06303 discloses a fusion proteinincluding amino acid sequences of HIV Vif, Vpu and Nef, wherein thecomponent proteins are contiguous with another component protein orseparated by non-component proteins such as amino acid sequences, whichmake up proteolytic cleavage sites. It is disclosed that it is preferredto use those fusion proteins that comprise proteolytic cleavage sitesbetween the component proteins. Since the component proteins areseparated by proteolytic cleavage sites native HIV proteins are producedthat are known to be harmful. To reduce any harmful effects of the HIVproteins that result from the cleavage of the fusion protein WO 02/06303suggests using attenuated proteins. Thus, WO 02/06303 teaches to use afusion protein comprising the HIV Vif, Vpr and Nef protein, whereincleavage sites are inserted between the HIV proteins and wherein the HIVproteins are attenuated proteins. However, the disadvantage ofattenuated proteins is that the amino acid sequence of the attenuatedprotein differs from the amino acid sequence of the native protein sothat an immunization with the attenuated protein may lead to an immuneresponse that only weakly recognizes the native protein or that evendoes not recognize the native protein at all.

OBJECT OF THE INVENTION

It was the object of the present invention to provide a vaccine allowingthe generation of an effective immune response, in particular aneffective cytotoxic T cell response, against several or allregulatory/accessory proteins of HIV, wherein the regulatory/accessoryHIV proteins in the vaccine or produced by the vaccine are lessfunctional than the native, individual regulatory/accessory proteins sothat the risk is reduced that the accessory/regulatory proteins in thevaccine exert undesired side effects and wherein the less active HIVproteins induce a similar immune response than the native HIV proteins.

DETAILED DESCRIPTION OF THE INVENTION

This object has been achieved by the provision of a fusion proteincomprising the amino acid sequence of at least four different HIVproteins selected from Vif, Vpr, Vpu, Vpx, Rev, Tat and Nef orderivatives of the amino acid sequence of one or more of said proteins,wherein the fusion protein is not processed to individual HIV proteinshaving the natural N and C termini. In particular the object of thepresent invention has been achieved by nucleic acids and vectorsencoding said fusion proteins.

If the fusion protein is produced in animal cells, including humancells, the fusion protein is not cleaved by cellular proteases in such away that accessory/regulatory proteins with native N- and C-termini areobtained.

Due to the fact that an HIV protein that is part of a fusion protein hasan altered secondary/tertiary structure compared to the individual HIVprotein, the HIV protein in the fusion protein is less functional thanthe individual protein, if not fully dysfunctional. Aregulatory/accessory protein that is less functional or even notfunctional at all does not have the undesired side effects of the HIVprotein in its native conformation. As far as the immunogenicity isconcerned there is no substantial difference when the immunogenicity ofthe fusion protein is compared with the immunogenicity of the individualHIV regulatory/accessory proteins that form the fusion protein. Inparticular there is no substantial difference with respect to thecytotoxic T cell (CTL) response since the epitopes that are presented tothe immune system are identical. The same considerations also apply ifthe fusion protein is administered to the patient.

In the context of the present invention the term “HIV” refers to any HIVgroup, subtype (clade), strain or isolate known to the person skilled inthe art. In particular, HIV may be HIV-1 or HIV-2. HIV-1 has beenclassified in nine subtypes (clades A through I), whereas HIV-2 has beenclassified in five subtypes (A through E), which are all covered by thescope of the present invention. The most preferred HIV clades accordingto the present invention are HIV-1 clades A, B and C. However, theinvention is not restricted to these most preferred clades.

The protein sequences of the HIV regulatory proteins Vif, Vpr, Vpu, Rev,Tat, Vpx and Nef are known to the person skilled in the art. By way ofexample and without being restricted to said embodiments reference ismade to the various sequences as disclosed in the genebank database, inparticular to the sequence of the HIV-1 isolate HXB2R having thegenebank accession number KO3455. In this genebank entry the sequencesof the various HIV1 genes and of the proteins encoded by said genes isspecified.

Preferably the HIV proteins that form the fusion protein are derivedfrom the same clade. According to an alternative embodiment the HIVproteins that form the fusion protein are derived from two or moreclades. It is also possible that one or more of the HIV proteins thatform the fusion protein are HIV-1 proteins and that one or more of theHIV proteins that form the fusion protein are HIV-2 proteins.

The amino acid sequences of the HIV proteins that form the fusionprotein are preferably sequences that are encoded by known HIV isolates,i.e. the amino acid sequence of the HIV proteins in the fusion proteinis identical to the amino acid sequences of the corresponding proteinsas encoded by naturally occurring HIV isolates. Alternatively the aminoacid sequence of one or more HIV proteins in the fusion protein may be aconsensus sequence, i.e. a sequence that as such may not be found in aknown HIV isolate but that shows an optimal homology—in particular withrespect to CTL-epitopes—to several or all known HIV isolates. Computeralgorithms to calculate a consensus sequence are known to the personskilled in the art.

In an alternative embodiment the fusion protein may comprise derivativesof the amino acid sequence of one or more HIV proteins that are part ofthe fusion protein. The term “derivative of the amino acid sequence ofan HIV protein” as used in the present specification refers to HIVproteins that have an altered amino acid sequence compared to thecorresponding naturally occurring HIV protein. An altered amino acidsequence may be a sequence in which one or more amino acids of thesequence of the HIV protein are substituted, inserted or deleted. Moreparticularly a “derivative of the amino acid sequence of an HIV protein”is an amino acid sequence showing a homology of at least 50%, morepreferably of at least 70%, even more preferably of at least 80%, mostpreferably of at least 90% when the corresponding part of the amino acidsequence in the fusion protein is compared to the amino acid sequence ofthe respective HIV protein of known HIV isolates. An amino acid sequenceis regarded as having the above indicated sequence homology even if thehomology is found for the corresponding protein of only one HIV isolate,irrespective of the fact that there might be corresponding proteins inother isolates showing a lower homology. By way of example, if a Vprderivative in the fusion protein shows a homology of 95% to the Vprsequence of one HIV isolate, but only a homology of 50-70% to (all)other HIV isolates, the homology of said Vpr derivative is regarded asbeing of at least 90%.

It has been pointed out above that the HIV proteins in the fusionprotein have a reduced activity, or even no activity at all, compared tothe individual proteins, since the conformation of the proteins in thefusion protein is different to the natural conformation of thebiologically active proteins. However, it might be desirable to furtherreduce the risk that the HIV proteins in the fusion protein arebiologically active. To this end particularly preferred “derivatives” ofan individual HIV protein that is part of a fusion protein are aminoacid sequence derivatives in which several amino acids are deleted,inserted or substituted, more preferably not more than 10 amino acids,most preferably not more than 5 amino acids to obtain an HIV proteinwith reduced activity or no activity at all. Tests are known to theperson skilled in the art how to determine whether an HIV protein hasreduced biological activity:

The molecular mechanism of the Vif protein, which is essential for viralreplication in vivo, remains unknown, but Vif possesses a strongtendency toward selfassociation. This multimerization was shown to beimportant for Vif function in viral life cycle (Yang S. et al., J BiolChem 2001; 276: 4889-4893). Additionally vif was shown to bespecifically associated with the viral nucleoprotein complex and thismight be functionally significant. (Khan M. A. et al., J Virol. 2001; 75(16): 7252-65). Thus, a vif protein with reduced activity shows areduced multimerization and/or assoziation to the nucleoprotein complex.

The Vpr protein plays an important role in the viral life cycle. Vprregulates the nuclear import of the viral preintegration complex andfacilitates infection of non dividing cells such as macrophages(Agostini et al., AIDS Res Hum Retroviruses 2002; 18(4):283-8).Additionally, it has transactivating activity mediated by interactionwith the LTR (Vanitharani R. et al., Virology 2001; 289 (2):334-42).Thus, a vpr with reduced activity shows decreased or even notransactivation and/or interaction with the viral preintegrationcomplex.

Vpx, which is highly homologous to Vpr, is also critical for efficientviral replication in non-dividing cells. Vpx is packaged in virusparticles via an interaction with the p6 domain of the gag precursorpolyprotein. Like Vpr Vpx is involved in the transportation of thepreintegration complex into the nucleus (Mahalingam et al., J. Virol2001; 75 (1):362-74). Thus, a Vpx with reduced activity has a decreasedability to associate to the preintegration complex via gag precurser.

The Vpu protein is known to interact with the cytoplasmic tail of theCD4 and causes CD4 degradation (Bour et al., Virology 1995; 69(3):1510-20). Therefore, Vpu with reduced activity has a reduced abilityto trigger CD4 degradation.

The relevant biological activity of the well-characterized Tat proteinis the transactivation of transcription via interaction with thetransactivation response element (TAR). It was demonstrated that Tat isable to transactivate heterologous promoters lacking HIV sequences otherthan TAR (Han P. et al., Nucleic Acid Res 1991; 19 (25):7225-9). Thus, atat protein with reduced activity shows reduced transactivation ofpromoters via the TAR element.

Nef protein is essential for viral replication responsible for diseaseprogression by inducing the cell surface downregulation of CD4 (Lou T etal., J Biomed Sci 1997;4(4):132). This downregulation is initiated bydirect interaction between CD4 and Nef (Preusser A. et al., BiochemBiophys Res Commun 2002;292 (3):734-40). Thus, Nef protein with reducedfunction shows reduced interaction with CD4.

The relevant function of Rev is the posttranscriptional transactivationinitiated by interaction with the Rev-response element (RRE) of viralRNA (Iwai et al., 1992; Nuceic Acids Res 1992; 20 (24):6465-72). Thus, aRev with reduced activity shows a reduced interaction with the RRE.

The fusion proteins according to the present invention comprise theamino acid sequence of at least four different HIV proteins selectedfrom Vif, Vpr, Vpu, Rev, Vpx, Tat and Nef. The fusion protein maypreferably comprise 5, 6 or all of said HIV proteins. The order of theHIV proteins in the fusion protein is not critical.

One or more of the at least four different HIV proteins may be comprisedin the fusion protein in two or more copies. Thus, by way of example afusion protein according to the present invention may comprise Vif, Vpr,Vpu and two copies of Rev. The amino acid sequence of the two or morecopies of a HIV protein may be identical. Alternatively, the amino acidsequence of the copies may be different, in particular if proteinsequences are used that are derived from different HIV strains or clades(e.g. one copy of an HIV-1 Rev and one copy of an HIV-2 Rev).

Adjacent HIV proteins in the fusion protein may be fused withoutadditional amino acids or fused in such a way that two adjacent HIVproteins in the fusion protein are separated by at least one additionalamino acid. Also combinations of both are within the scope of thepresent invention. By way of example, in a fusion protein according tothe present invention comprising the amino acid sequence of four HIVproteins two adjacent HIV proteins may be directly linked to each other,whereas the third and fourth HIV proteins are linked via additionalamino acids. The term “additional amino acid” in the context of thisembodiment refers to amino acids that are not found in this position inthe naturally occurring HIV proteins.

Thus, the fusion protein according to the present invention preferablyhas the following general formula:+P1−−−P2−−−P3−−−P4−−−P5*−−−P6*−−−P7*+wherein P1 to P7 are different HIV proteins selected from Vif, Vpr, Vpx,Vpu, Tat, Rev and Nef, wherein the fusion protein comprises at leastfour different of said HIV proteins, i.e. P1 to P4 and optionally one(P5*), two (P5*−−−P6*) or three (P5*−−−P6*−−−P7*) additional of said HIVproteins. The abbreviation “−−−” independently stands for 0 to nadditional amino acids. When “−−−” stand for 0 amino acids, the adjacentHIV proteins are directly fused to each other without additional aminoacids. When “−−−” stands for 1 to n amino acids the adjacent HIVproteins are separated by one to n amino acids. The upper limit of theadditional amino acids, i.e. the integer n, depends on the maximal sizeof the fusion protein that can be produced or expressed in cells.

According to one embodiment all “−−−” stand independently for 0 to 20,more preferably 0 to 10, even more preferably 0 to 5 additional aminoacids.

According to an alternative embodiment at least one of “−−−” stands forthe amino acid sequence of an additional protein or a part thereof,which is not an HIV protein selected from Vif, Vpr, Vpx, Vpu, Rev, Tatand Nef. Thus, according to this alternative embodiment the additionalprotein is flanked by regulatory/accessory HIV proteins. The additionalprotein may be any protein. More preferably the additional proteincomprises additional epitopes that may help to induce a better immuneresponse against HIV. Thus, the additional protein may be the HIV Env,Gag and/or Pol protein or parts thereof. In this context the term “part”of Env, Gag and Pol refers to an amino acid stretch derived from one ofsaid protein, which comprises at least one epitope. More preferably theterm part refers to at least 10, even more preferably to at least 20,most preferably to at least 50 amino acids from one of said proteins.According to an related embodiment at least one of “−−−” stands for theamino acid sequence of one or more of the proteins P1 to P7 that arepart of the fusion protein. Thus, in this case the fusion protein maycomprise one or more copies of one or more of the proteins that are partof the fusion protein. As pointed out the copies of the proteins may ormay not have an identical amino acid sequence.

In the above formula the abbreviation “+” independently stands for 0 ton additional terminal amino acid. Thus, the fusion protein according tothe present invention may or may not comprise additional amino acids atthe C and/or N-terminus of the protein. According to one embodiment atleast one of “+” stands for the amino acid sequence of an additionalprotein or part thereof, which is not an HIV protein selected from Vif,Vpr, Vpx, Vpu, Rev, Tat and Nef. Thus, according to this embodiment thefusion protein comprises at its C and/or N terminus an additionalprotein, which is not Vif, Vpr, Vpx, Vpu, Rev, Tat or Nef. Theadditional protein may be any protein. More preferably the additionalprotein comprises additional epitopes that may help to induce a betterimmune response against HIV. E.g., the additional protein may be the HIVEnv, Gag and/or Pol protein or parts thereof. In this context the term“part” of Env, Gag and Pol refers to an amino acid stretch derived fromone of said protein, which comprises at least one epitope. Morepreferably the term part refers to at least 10, even more preferably toat least 20, most preferably to at least 50 amino acids from one of saidproteins.

According to an alternative embodiment at least one of “+” stands for anamino acid sequence that allows the easy detection or purification ofthe fusion protein. Thus, at least one of “+” might for example be a tagsuch as a His tag.

According to the present invention the fusion protein is not processedto individual HIV proteins having the natural N- and C-termini. Moreparticularly, the fusion protein according to the present invention isnot processed to individual HIV proteins having the natural N- andC-termini, when expressed in human cells. Methods are known to theperson skilled in the art how to check whether a fusion protein whenexpressed in human cells is processed to individual HIV proteins havingthe natural N- and C-termini. In this context reference is made toAyyavoo et al., AIDS 2000, 14, 1-9, in particular to the experimentdisclosed in FIG. 2 of said publication. Briefly, the person skilled inthe art might easily express the respective fusion protein in humancells such as HeLa cells; the cells are then lysed and the cell lysatesare subjected to Western blotting experiments or immunoprecipitationassays with antibodies specific for the individual HIV proteins thattogether form the respective HIV fusion protein. For a fusion proteinaccording to the present invention no significant amount of HIV proteinsis detected the size of which corresponds to the size of an individualHIV regulatory/accessory protein.

In order to ensure that the fusion protein according to the presentinvention is not processed to individual HIV proteins having the naturalN- and C-termini, the fusion protein should not contain specificcleavage sequences for cellular proteases, which might trigger thegeneration of HIV proteins having the natural N- and C-termini, betweenthe amino acid sequences of the HIV proteins that form the fusionprotein. Thus, the amino acid sequence “−−−” as abbreviated in the abovegeneral formula does not contain specific cleavage sequences forcellular proteases, which might trigger the generation of HIV proteinshaving the natural N- and C-termini. In particular the fusion proteindoes not contain the cleavage sequence REKRAVVG (one letter amino acidcode) between the amino acid sequences of the different HIV proteinsthat form the fusion protein. Further cleavage sequences for cellularproteases are known to the person skilled in the art. Thus, the personskilled in the art can easily avoid to include cleavage sequences for(cellular) proteases that might lead to individual HIV proteins havingnatural N- and C-termini. An example for the cleavage sequence of acysteinprotease is Ile/leu-X-Thr-X-Gly.

The proteins according to the present invention do not comprise specificcleavage sequences leading to HIV proteins having both, the native N-and C-termini. However, this does not generally exclude the presence ofcleavage sites for cellular proteases between the proteins in the fusionprotein as long as these cleavage sites do not mediate the generation ofHIV proteins having both, a natural N-terminus and a natural C-terminus.In particular, the amino acid sequence “−−−” as abbreviated in the abovegeneral formula may comprise cleavage sites for the proteases that areinvolved in the generation of short peptides presented on MHCI or MHCII.According to this embodiment the result of the cleavage reaction is ashort peptide stretch of preferably less than 20 amino acids, the N- orC-terminus of which may correspond to the N- or C-terminus of one of theHIV accessory/regulatory proteins. However, these short peptides, whenproduced during the process of presentation of antigens, do not haveanymore the activity of the HIV protein from which they are derived.

The invention further relates to nucleic acids encoding the abovedefined fusion proteins according to the present invention. The nucleicacid may be DNA or RNA. Preferably the nucleic acid is DNA if it isintended to insert the nucleic acid into human cells by using a DNAvector such as a plasmid or a vector based on a DNA virus.

Methods are known to the person skilled in the art how to construct anucleic acid encoding the fusion protein according to the presentinvention. Without being bound to the following methods, the personskilled in the art may start from a genomic HIV clone, from a subgenomicHIV clone or from any starting material, such as plasmids, comprisingthe coding sequence of one or more of the regulatory/accessory HIVproteins. If the coding sequence of a regulatory/accessory protein is inthe form of a continuous reading frame, said coding sequence may beisolated by cleaving the nucleic acid comprising said coding sequencewith appropriate restriction enzymes. The thus obtained DNA fragmentsmay be used for further cloning. Alternatively the coding sequences ofan accessory/regulatory protein may be obtained by using PolymeraseChain Reaction (PCR) methods with appropriate primers. If theregulatory/accessory proteins are encoded by more than one exon, as itis the case e.g. for Tat and Rev, it may be necessary to independentlyclone the different exons and to fuse them to generate a continuousreading frame for the regulatory/accessory protein or to use reversetranscription technology such as RT-PCR.

A coding sequence can also be provided by gene synthesis, i.e. bygenerating a gene using a set of complementary and/or overlappingoligonucleotides.

In order to obtain a fusion protein the nucleic acid encoding saidfusion protein preferably contains a continuous reading frame.Consequently, the stop codons of all but the last sequence encoding HIVproteins or additional proteins are preferably mutated into a codoncoding for an amino acid or deleted completely. Preferably, this can beeasily achieved if for PCR specific primers are used that amplify thecoding sequence without the stop codon. In other words, according tothis alternative the downstream primer should not be complementary tothe stop codon. The amplified DNA fragment therefore will not contain astop codon and can be cloned into the cloning vector. Alternatively, itis also possible to clone a coding sequence with its stop codon into thecloning vector. The stop codon can be deleted later, e.g. by usingspecific endonucleases or by mutagenization.

The result of the cloning steps should be a continuous reading frameencoding the fusion protein according to the present invention.

The regulatory elements that are necessary to obtain the expression ofthe fusion protein may be any regulatory elements that drive theexpression in the desired expression system. If it is intended toproduce the fusion protein in prokaryotic cells such as Escherichia coliit is preferable to use a bacterial or phage promoter. If it is intendedto express the fusion protein in eukaryotic cells it is preferable touse an eukaryotic or viral promoter/enhancer. If it is intended toexpress the fusion protein by using a poxviral promoter (see below) itis preferable to use a poxviral promoter such as the 7.5 promoter or theATI promoter.

As pointed out above the fusion protein may comprise fusion partnerswhich are not HIV proteins selected from Vif, Vpr, Vpx, Vpu, Tat, Revand Nef. Thus, the fusion protein may comprise the amino acid sequenceof other proteins or parts thereof. Examples of other proteins are theHIV Gag, Pol and Env proteins. Consequently, the nucleic acid accordingto the present invention may comprise also the coding sequences for oneor more additional proteins or part thereof in the open reading frameencoding at least four regulatory/accessory HIV proteins or derivativesthereof.

In a further embodiment of the present invention the nucleic acid mayfurther comprise independent expression cassettes encoding additionalproteins that may help to further improve the immune response againstHIV. In a preferred embodiment the nucleic acid may further compriseexpression cassettes comprising the coding sequence of at least oneadditional HIV protein selected from Gag, Pol and Env or parts thereof.Even more preferably the nucleic acid may comprise in addition to thecoding sequence of the fusion protein the coding sequences of all HIVproteins Gag, Pol and Env. The nucleic acid is preferably part of avector. The nucleic acid may also be the viral genome or part thereof ofa viral vector, preferably a poxvirus vector such as MVA. Thus, it ispossible to express from the poxviral vector the fusion protein as wellas the additional HIV proteins, e.g. at least one additional HIV proteinselected from Gag, Pol and Env.

The invention further relates to vectors comprising a nucleic acidaccording to the present invention. The term “vector” refers to anyvectors known to the person skilled in the art. A vector can be aplasmid vector such as pBR322 or a vector of the pUC series. Morepreferably the vector is a virus vector. In the context of the presentinvention the term “viral vector” or “virus vector” refers to aninfectious and/or attenuated virus comprising a viral genome. In thiscase the nucleic acid of the present invention is part of the viralgenome of the respective viral vector and/or constitutes the viralgenome. The recombinant vectors can be used for the infection of cellsand cell lines, in particular for the infection of living animalsincluding humans. Typical virus vectors according to the presentinvention are adenoviral vectors, retroviral vectors or vectors on thebasis of the adeno associated virus 2 (MV2). Most preferred are poxviralvectors. The poxvirus may be preferably a canarypox virus, afowlpoxvirus or a vaccinia virus. More preferred is modified vacciniavirus Ankara (MVA) (Sutter, G. et al. [1994], Vaccine 12: 1032-40). Atypical MVA strain is MVA 575 that has been deposited at the EuropeanCollection of Animal Cell Cultures under the deposition number ECACCV00120707. Most preferred is MVA-BN or a derivative thereof, which hasbeen described in the PCT application PCT/EP01/13628 filed at theEuropean Patent Office on Nov. 22, 2001, entitled “Modified VacciniaAnkara Virus Variant”. MVA-BN has been deposited at the EuropeanCollection of Animal Cell Cultures with the deposition number ECACCV00083008. By using MVA-BN or a derivative thereof the additionaltechnical problem has been solved to provide a particular safe virusvaccine against HIV since the MVA-BN virus vector is an extremelyattenuated virus, which is derived from Modified Vaccinia Ankara virusand which is characterized by the loss of its capability toreproductively replicate in human cells. MVA-BN is safer than any otherknown vaccinia virus strains due to a lack of replication in humans. Ina preferred embodiment the invention concerns as a viral vectorcontaining the DNA according to the present invention MVA-BN andderivatives of MVA-BN. The features of MVA-BN, the description ofbiological assays allowing to evaluate whether a MVA is MVA-BN or aderivative thereof and methods allowing to obtain MVA-BN or a derivativethereof are disclosed in the above referenced PCT applicationPCT/EP01/13628, which is herewith incorporated by reference.

Thus, according to these embodiments the invention concerns preferably arecombinant MVA, such as MVA-BN, comprising in the viral genome anexpression cassette encoding a fusion protein according to the presentinvention.

Methods to insert the nucleic acid according to the present inventioninto the viral genome and methods to obtain recombinant viruses areknown to the person skilled in the art.

In a recombinant vaccinia virus the expression of the DNA according tothe present invention is preferably, but not exclusively, under thetranscriptional control of a poxvirus promoter, more preferably of avaccinia virus promoter. The insertion of the DNA according to thepresent invention is preferably into a non-essential region of the virusgenome. In another preferred embodiment of the invention, theheterologous nucleic acid sequence is inserted at a naturally occurringdeletion site of the poxviral genome (disclosed in PCT/EP96/02926).However, the nature of the insertion site is not critical for thepresent invention as long as a recombinant Vaccinia virus is obtained.Thus, the person skilled in the art may easily envisage further suitableinsertion sites.

Preferably the viral vector, in particular the poxviral vector maycomprise additional retroviral genes selected from HIV Gag, Pol and Envgenes in the viral genome, in addition to the coding sequence for thefusion protein according to the present invention. More preferably theviral vector, in particular the poxviral vector, may comprise all HIVgenes encoding Gag, Pol and Env in addition to the coding sequence forthe fusion protein according to the present invention. These additionalgenes might have been inserted with the same nucleic acid according tothe present invention. According to this embodiment all HIV genes wouldbe located in the same insertion site in the viral genome. In analternative embodiment the additional genes are inserted in differentlocations of the viral genome.

In a preferred embodiment the present invention concerns the nucleicacid, the vector or the fusion protein according to the presentinvention as a vaccine for the at least partial prophylaxis against HIVinfections and AIDS. A “vaccine” is a compound, i.e. a nucleic acid, afusion protein, a vector or a virus that induces a specific immuneresponse.

According to one alternative of this embodiment the “vaccine” accordingto the present invention is based on the fusion protein according to thepresent invention.

In a preferred embodiment the nucleic acid according to the presentinvention, in particular DNA, is used as a vaccine. It is known by theperson skilled in the art that the administration of naked DNA harboringa eukaryotic expression cassette as in the present invention, inparticular the intramuscular injection of DNA leads to the expression ofthe protein encoded by the expression cassette. The protein is exposedto the immune system and a specific immune response is raised.

In an alternative embodiment the vaccination is made by administering avector according to the present invention, in particular a viral vector,more preferably a poxvirus vector, most preferably a vaccinia virusvector, e.g. a MVA vector.

For the preparation of a vaccinia virus based vaccine, the virusaccording to the invention is converted into a physiologicallyacceptable form. This can be done based on the experience in thepreparation of poxvirus vaccines used for vaccination against smallpox(as described by Stickl, H. et al. [1974] Dtsch. med. Wschr. 99,2386-2392). For example, the purified virus is stored at −80° C. with atiter of 5×10⁸ TCID₅₀/ml formulated in about 10 mM Tris, 140 mM NaCl pH7.4. For the preparation of vaccine shots, e.g., 10²-10⁸ particles ofthe virus are lyophilized in 100 ml of phosphate-buffered saline (PBS)in the presence of 2% peptone and 1% human albumin in an ampoule,preferably a glass ampoule. Alternatively, the vaccine shots can beproduced by stepwise freeze-drying of the virus in a formulation. Thisformulation can contain additional additives such as mannitol, dextran,sugar, glycine, lactose or polyvinylpyrrolidone or other additives suchas antioxidants or inert gas, stabilizers or recombinant proteins (e.g.human serum albumin) suitable for in vivo administration. The glassampoule is then sealed and can be stored between 4° C. and roomtemperature for several months. However, as long as no need exists theampoule is stored preferably at temperatures below −20° C. Forvaccination the lyophilisate can be dissolved in 0.1 to 0.5 ml of anaqueous solution, preferably physiological saline or Tris buffer, andadministered either systemically or locally, i.e. by parenterally,intramuscularly or any other path of administration know to the skilledpractitioner. The mode of administration, the dose and the number ofadministrations can be optimized by those skilled in the art in a knownmanner. Most preferred for poxvirus vectors is subcutaneous orintramuscular administration.

If the vaccine is a MVA-BN vector or derivative thereof comprising a DNAaccording to the present invention, a particular embodiment of thepresent invention concerns the administration of the vaccine intherapeutically effective amounts in a first inoculation (“priminginoculation”) and in a second inoculation (“boosting inoculation”).

If the vaccine is a MVA-BN vector or derivative thereof comprising a DNAaccording to the present invention a particular embodiment of thepresent invention concerns a kit for vaccination comprising a MVA-BNvirus vector according to the present invention for the firstvaccination (“priming”) in a first vial/container and for a secondvaccination (“boosting”) in a second vial/container.

Thus the invention concerns in the vaccine embodiments a vaccinecomprising a nucleic acid, a vector or a fusion protein according to thepresent invention and the use of said nucleic acid, vector or proteinfor the preparation of a vaccine.

According to a further embodiment the invention concerns a method forprotecting an animal, including a human, against an HIV infection byadministering to an animal, including a human, in need thereof a fusionprotein according to the present invention, a nucleic acid according tothe present invention or a vector according to the present invention.

Moreover, the invention concerns a method of producing a proteinaccording to the present invention, comprising the steps of (i)transfecting a host cell with a nucleic acid or a vector according tothe present invention or (ii) infecting a host cell with a viral vectoraccording to the present invention, (iii) expressing the fusion proteinin the transfected host cell of step (i) or the infected host cell ofstep (ii), and (iv) recovering the fusion protein.

The invention further relates to host cells transfected with a nucleicacid or a vector according to the present invention or infected with aviral vector according to the present invention.

According to an alternative embodiment the fusion protein may compriseat least three different HIV proteins selected from Vif, Vpr, Vpu, Rev,Vpx and Tat. The fusion protein may preferably comprise 4, 5 or all ofsaid HIV proteins. A typical fusion protein according to this embodimentcomprises the amino acid sequence of the HIV proteins Vpr, Vif, Vpu, Revand Tat or derivatives of the amino acid sequence of one or more of saidproteins. As pointed out above, the order of the HIV proteins in thefusion protein is not critical. All preferred embodiments as specifiedabove also apply for this alternative embodiment.

SHORT DESCRIPTION OF THE FIGURES

FIG. 1: Schematic presentation of Annealing of Oligonucleotides

The picture shows the annealing of four Oligonucleotides. They aresingle stranded and can be annealed by complementary ends. The gaps arefilled in with a polymerase, which exhibits a proofreading activity (egPfx polymerase).

FIG. 2: Schematic presentation of annealing of four genes of the blob

The vif gene shows a overlapping sequence with the vpr fragment, the vpucoding fragment shows an overlapping sequence with the rev gene (grey).The PCR fragments are denatured and the overlapping complementary endsare hybridized. The resulting gaps are filled using Pfx polymerase. Thevif-vpr fragment is fused to a overlapping sequence of the vpu-revfragment, which again is used for fusion.

FIG. 3: Cloning strategy of the sequence encoding a fusion proteinaccording to the present invention in a recombination vector forinsertion of foreign genes into the MVA genome

The fused vif, vpr, vpu and rev polyprotein coding region was amplifiedwith primers comprising a ClaI and ApaI restriction site. This pCRproduct was cloned into the ClaI/ApaI cutted vector pBNX65, whichcontains the Poxvirus ATI promoter. The tat coding region was amplifiedby PCR with primers containing an Acc65I restriction site and ligated tothe Acc65I linearized pBNX65+vif-rev. The resulting expression cassette(ATI promoter+sequence encoding a fusion protein according to thepresent invention) was isolated by PacI restriction and inserted in therecombination vector for insertion of foreign genes in the MVA genomeI4L intergenic region (pBNX39). PBNX39 contains sequences homologous tothe flanking sequences of the insertion site of the MVA genome (F1 I4Land F2 I4L). For selection of recombinant viruses after homologousrecombination of the MVA genome and pBNX39 the vector additionallycontains the E. coli gpt gene (phosphoribosyltransferase gene). Afterpurification of recombinant viruses, the selection cassette is deletedby homologous recombination between Flank 1 and a repeat sequence offlank 1 (F1rpt).

FIG. 4: Schematic presentation of the MVA genome

MVA contains a linear genome, which shows characteristic fragments afterrestriction with Hind III (A-O). The non functional region between theI4L and the I5L genes is located in the I fragment. Insertion of foreigngenes using pBNX39 occurs at position 56767-56768.

EXAMPLES Generation of a DNA Encoding a HIV Vif-Vpr-Vpu-Rev-Tat FusionProtein

The single genes of the HIV genome were either prepared by PCR out ofgenomic DNA by using standard PCR protocols or synthetically by atechnique, which is based on the annealing of oligonucleotides viaoverlapping sequences and fill in of the resulting single stranded gaps.

For the oligonucleotide based generation of coding regions of genes,which are to be inserted into the nucleic acid encoding the fusionprotein according to the present invention, 40 mer oligonucleotides with15 bp overlaps were designed. The sequence of the oligonucleotides isbased on the genomic map of the HIV1 isolate HXB2R that is derived fromstrain IIIB. The oligonucleotides for the annealing reaction or the PCRfor isolation of the required sequence were designed in that way, thatin the resulting coding region the stop codons for translationtermination were deleted. The tat gene was synthesized using oligoscontaining a Stop codon as this gene was to be inserted at the lastpostion of the nucleic acid encoding the fusion protein according to thepresent invention and therefore should contain a stop triplet for acorrect termination of translation of the polyprotein.

For the oligoannealing rection 10 cycles of a two step Pfx polymerase(Gibco-BRL) reaction (denaturation at 95° C. and annealing/extension at68° C.) were performed. During that reaction the overlapping sequencesof the oligos become annealed and the gaps are filled in by Pfxproofreading polymerase (FIG. 1).

For synthesis of the vif coding region, the first encoded gene in thenucleotide sequence encoding the fusion protein, a PCR using genomic HIVcDNA was performed. The PCR was performed in that way, that the vifcoding region was fused to the first 15 bp of the following vpr gene forthe subsequent annealing of vif and vpr. The Vpr coding region, whichcovers bp 5559-5847 of the HIV HXB2R genome, was prepared by annealingof 10 oligonucleotides. The resulting gaps were filled and aftersubsequent PCR for amplification the product contained the vpr codingregion fused to flanking regions for vif and vpu, which was to beinserted after vpr coding region.

The Vpu coding region was amplified by PCR out of the same cDNA used forsynthesis of vif and the resulting product contained the flankingregions for fusion with vpr and rev.

The rev coding region was synthesized by annealing of 14oligonucleotides, which cover the region bp 5970-6045 and 8379-8650 ofthe HIV HXB2R genome and 15 bp overlaps for annealing with vpu and tat.

The tat coding region was created by using 10 oligonucleotides, whichcover bp 5831-6045 and 8379-8466 of of the HIV HXB2R genome.

The vif and the vpr coding region as well as the vpu and the rev codingregion were fused by annealing of the two fragments via their overlapswith a two step Pfx polymerase reaction (FIG. 2). After additional PCRamplification of the fusion products, the fragments were purified andligated to each other via the overlap of vpr and vpu (FIG. 2). After PCRamplification of the resulting product (coding sequences forvif-vpr-vpu-rev) the tat coding region was fused by cloning of thevif-vpr-vpu-rev fragment and tat in adjacent cloning sites in apBluescriptKS+ vector containing the poxvirus ATI promoter (FIG. 3,pBNX65). The complete expression cassette was then isolated by PacIrestriction and inserted in pBNX39 (FIG. 3). PBNX39 contains sequenceshomologous to the MVA genome, which allows insertion in a non codingregion (I4L) of the genome (FIG. 4) by homologous recombination.

1. Nucleic acid encoding a fusion protein comprising an amino acidsequence of at least four non-attenuated HIV regulatory and accessoryproteins selected from the group consisting of Vif, Vpr, Vpu, Vpx, Rev,Tat, and Nef, wherein the fusion protein does not contain a specificcleavage sequence for a cellular protease.
 2. Nucleic acid according toclaim 1, wherein the nucleic acid is DNA.
 3. Nucleic acid according toclaim 2, wherein the expression of the fusion protein from the DNA iscontrolled by regulatory elements selected from eukaryotic, procaryoticand viral promoters.
 4. Nucleic acid according to claim 3, wherein theviral promoter is a poxviral promoter.
 5. Nucleic acid according toclaim 1, wherein the nucleic acid further comprises the coding sequencefor at least one additional HIV protein selected from the groupconsisting of Gag, Pol and Env.
 6. Nucleic acid according to claim 5,wherein the nucleic acid comprises the coding sequence for the HIV Gag,Pol and Env proteins.
 7. Nucleic acid according to claim 1 encoding afusion protein selected from the group consisting of Vif-Vpr-Vpu-Rev andVif-Vpr-Vpu-Rev-Tat.
 8. Nucleic acid according to claim 1 encoding afusion protein Vif-Vpr-Vpu-Rev.
 9. Vector comprising a nucleic acidaccording to claim
 1. 10. Vector comprising a nucleic acid according toclaim
 8. 11. Vector according to claim 9, wherein the vector is a viralvector.
 12. Vector according to claim 10 wherein the viral vector is aVaccinia virus vector.
 13. Vector according to claim 12, wherein theVaccinia virus vector is Modified Vaccinia Virus Ankara (MVA). 14.Vector according to claim 13, wherein MVA is selected from MVA-575deposited at the European Collection of Animal Cell Cultures (ECACC)under the deposition number VO01-20707 and MVA-BN deposited at the ECACCunder the deposition number V00083008.
 15. Method of producing a fusionprotein, comprising an amino acid sequence of at least fournon-attenuated HIV proteins selected from the group consisting of Vif,Vpr, Vpu, Vpx, Rev, Tat, and Nef, wherein the fusion protein does notcontain a specific cleavage sequence for a cellular protease, comprisingthe steps of transfecting a host cell with a nucleic acid according toclaim 1, expressing the fusion protein in the transfected host cell, andrecovering the fusion protein.
 16. Host cell transfected with a nucleicacid according to claim
 1. 17. Method of producing a fusion proteincomprising an amino acid sequence of at least four non-attenuated HIVproteins selected from the group consisting of Vif, Vpr, Vpu, Vpx, Rev,Tat, and Nef, wherein the fusion protein does not contain a specificcleavage sequence for a cellular protease, comprising the steps of:infecting a host cell with a vector according to claim 9, expressing thefusion protein in the infected host cell, and recovering the fusionprotein.
 18. Host cell infected with a vector according to claim
 9. 19.Method of producing a fusion protein comprising an amino acid sequenceof at least four non-attenuated HIV proteins selected from the groupconsisting of Vif, Vpr, Vpu, Vpx, Rev, Tat, and Nef, wherein the fusionprotein does not contain a specific cleavage sequence for a cellularprotease comprising the steps of transfecting a host cell with a viralvector according to claim 10, expressing the fusion protein in thetransfected host cell, and recovering the fusion protein.
 20. Host celltransfected with a viral vector according to claim
 11. 21. Nucleic acidencoding a fusion protein comprising an amino acid sequence of at leastfour regulatory/accessory HIV proteins selected from the groupconsisting of Vif, Vpr, Vpu, Vpx, Rev, Tat, and Nef, wherein the fusionprotein does not contain a specific cleavage sequence for a cellularprotease, and where there is no substantial difference when theimmunogenecity of the fusion protein is compared with the immunogenecityof the individual HIV regulatory/accessory proteins that form the fusionprotein.
 22. Nucleic acid according to claim 21, wherein the nucleicacid is DNA.
 23. Nucleic acid according to claim 22, wherein theexpression of the fusion protein from the DNA is controlled byregulatory elements selected from eukaryotic, procaryotic and viralpromoters.
 24. Nucleic acid according to claim 23, wherein the viralpromoter is a poxviral promoter.
 25. Nucleic acid according to claim 21,wherein the nucleic acid further comprises the coding sequence for atleast one additional HIV protein selected from the group consisting ofGag, Pol and Env.
 26. Nucleic acid according to claim 25, wherein thenucleic acid comprises the coding sequence for the HIV Gag, Pol and Envproteins.
 27. Nucleic acid according to claim 21 encoding a fusionprotein selected from the group consisting of Vif-Vpr-Vpu-Rev andVif-Vpr-Vpu-Rev-Tat.
 28. Nucleic acid according to claim 21 encoding afusion protein Vif-Vpr-Vpu-Rev.
 29. Vector comprising a nucleic acidaccording to claim
 21. 30. Vector comprising a nucleic acid according toclaim
 28. 31. Vector according to claim 29, wherein the vector is aviral vector.
 32. Vector according to claim 30, wherein the viral vectoris a Vaccinia Virus vector.
 33. Vector according to claim 31, whereinthe Vaccinia virus vector is Modified Vaccinia Virus Ankara (MVA). 34.Vector according to claim 33, wherein MVA is selected from MVA-575deposited at the European Collection of Animal Cell Cultures (ECACC)under the deposition number VO01-20707 and MVA-BN deposited at the ECACCunder the deposition number V00083008.
 35. Method of producing a fusionprotein, comprising an amino acid sequence of at least fourregulatory/accessory HIV proteins selected from the group consisting ofVif, Vpr, Vpu, Vpx, Rev, Tat, and Nef, wherein the fusion protein doesnot contain a specific cleavage sequence for a cellular protease, andwhere there is no substantial difference when the immunogenecity of thefusion protein is compared with the immunogenecity of the individual HIVregulatory/accessory proteins that form the fusion protein, comprisingthe steps of transfecting a host cell with a nucleic acid according toclaim 21 expressing the fusion protein in the transfected host cell, andrecovering the fusion protein.
 36. Host cell transfected with a nucleicacid according to claim
 21. 37. Method of producing a fusion proteincomprising an amino acid sequence of at least four HIV proteins selectedfrom the group consisting of Vif, Vpr, Vpu, Vpx, Rev, Tat, and Nef,wherein the fusion protein does not contain a specific cleavage sequencefor a cellular protease, wherein the amino acid sequence of an HIVprotein in the fusion protein shows a homology of at least 90% whencompared with the amino acid sequence of the respective HIV protein ofknown isolates, and where there is no substantial difference when theimmunogenecity of the fusion protein is compared with the immunogenecityof the individual HIV regulatory/accessory proteins that form the fusionprotein, comprising the steps of: infecting a host cell with a vectoraccording to claim 29, expressing the fusion protein in the infectedhost cell, and recovering the fusion protein.
 38. Host cell infectedwith a vector according to claim
 29. 39. Method of producing a fusionprotein comprising an amino acid sequence of at least four HIV proteinsselected from the group consisting of Vif, Vpr, Vpu, Vpx, Rev, Tat, andNef, wherein the fusion protein does not contain a specific cleavagesequence for a cellular protease, and where there is no substantialdifference when the immunogenecity of the fusion protein is comprisingthe steps of transfecting a host cell with a viral vector according toclaim 31, expressing the fusion protein in the transfected host cell,and recovering the fusion protein.
 40. Host cell infected with a vectoraccording to claim 31.